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(2009).21. T. Y. Tsai et al., Science 321, 126–129 (2008).22. J. K. Kim, D. B. Forger, Mol. Syst. Biol. 8, 630

(2012).23. B. Novák, J. J. Tyson, Nat. Rev. Mol. Cell Biol. 9, 981–991

(2008).24. E. L. O’Brien, E. Van Itallie, M. R. Bennett, Math. Biosci. 236,

1–15 (2012).25. F. Naqib et al., Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 85,

046210 (2012).26. W. H. Mather, N. A. Cookson, J. Hasty, L. S. Tsimring,

R. J. Williams, Biophys. J. 99, 3172–3181 (2010).27. A. Prindle et al., Nature 508, 387–391 (2014).

28. H. Cho et al., Nature 485, 123–127 (2012).29. W. Mather, M. R. Bennett, J. Hasty, L. S. Tsimring, Phys. Rev.

Lett. 102, 068105 (2009).

ACKNOWLEDGMENTS

This work was funded by the National Institutes of Health, throughthe joint NSF–National Institute of General Medical SciencesMathematical Biology Program grant R01GM104974 (M.R.B. andK.J.), the Robert A. Welch Foundation grant C-1729 (M.R.B.),the Hamill Foundation (M.R.B.), NSF grant DMS-0931642 to theMathematical Biosciences Institute (J.K.K.), and the ChinaScholarship Council (Y.C.). M.R.B., Y.C., K.J., and J.K.K. conceivedand designed the study. Y.C. performed the experiments andanalyzed the data. A.J.H. designed and fabricated the microfluidicdevices. J.K.K. performed the computational modeling and

analyzed simulations. M.R.B. supervised the project. All authorswrote the manuscript. The mathematical model and experimentaldata are archived in the BioModels Database at www.ebi.ac.uk/biomodels-main/MODEL1505050000.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/349/6251/986/suppl/DC1Materials and MethodsFigs. S1 to S10Table S1Movie S1References (30–52)

26 November 2014; accepted 2 July 201510.1126/science.aaa3794

MUCOSAL IMMUNOLOGY

The microbiota regulates type 2immunity through RORgt+ T cellsCaspar Ohnmacht,1*† Joo-Hong Park,1* Sascha Cording,1 James B. Wing,2

Koji Atarashi,3,4 Yuuki Obata,5 Valérie Gaboriau-Routhiau,6,7,8 Rute Marques,1‡Sophie Dulauroy,1 Maria Fedoseeva,9 Meinrad Busslinger,10 Nadine Cerf-Bensussan,6,7

Ivo G. Boneca,11,12 David Voehringer,13 Koji Hase,5 Kenya Honda,3,14

Shimon Sakaguchi,2,15 Gérard Eberl1§

Changes to the symbiotic microbiota early in life, or the absence of it, can lead toexacerbated type 2 immunity and allergic inflammations. Although it is unclear howthe microbiota regulates type 2 immunity, it is a strong inducer of proinflammatoryT helper 17 (TH17) cells and regulatory T cells (Tregs) in the intestine. Here, we reportthat microbiota-induced Tregs express the nuclear hormone receptor RORgt anddifferentiate along a pathway that also leads to TH17 cells. In the absence of RORgt+

Tregs, TH2-driven defense against helminths is more efficient, whereas TH2-associatedpathology is exacerbated. Thus, the microbiota regulates type 2 responses through theinduction of type 3 RORgt+ Tregs and TH17 cells and acts as a key factor in balancingimmune responses at mucosal surfaces.

Allergic reactions are on the rise in indus-trialized nations, paralleling a decrease inthe incidence of infectious diseases (1, 2).The hygiene hypothesis proposes that ex-posure to pathogens reduces the risk of

allergy, a notion that may be extended to ex-posure to the symbioticmicrobiota. In support ofthis hypothesis, germfree mice, devoid of micro-organisms, develop increased susceptibility toallergy (3–6). Furthermore, a developmental timewindow during childhood determines such sus-ceptibility (1, 2).Mice treated earlywith antibiotics,which deeply affect the microbiota, develop anincreased susceptibility to allergy (7) that can lastinto adulthood (8), an effect also found in micethat remain germfree until weaning (9).The mechanism by which the microbiota reg-

ulates type 2 responses remains unclear. A directeffect of microbiota on type 2 cells, such as Thelper 2 (TH) cells and innate lymphoid cells(ILC) 2, has not been documented. In contrast,symbionts are necessary for the differentiation ofTH17 cells that produce interleukin (IL)–17 andIL-22 (10), cytokines involved in homeostasis anddefense of mucosal surfaces, and a subset of in-testinal regulatory T cells (Tregs) (11). Intriguingly,the absence of extrathymically generated Tregs

leads to spontaneous type 2 pathologies at muco-sal sites (12). As intestinal Tregs recognize bacte-rial antigens (11), the microbiota may regulatetype 2 responses through the induction of extra-thymically generated Tregs.The nuclear hormone receptor RORgt is a key

transcription factor for the differentiation ofTH17 cells and ILC3s (13, 14). In addition, a sub-stantial fraction of RORgt+ T cells residing in thelamina propria of the small intestine does notexpress IL-17, but rather IL-10, the Treg markerFoxP3 (a transcription factor), and has regulatoryfunctions (15). Furthermore, the generation ofsuch RORgt+ Tregs requires the microbiota (16).Using reporter mice for the expression of RORgtand Foxp3, we found that a majority of RORgt+

T cells in the colon of adult mice expressed Foxp3,and, reciprocally, a majority of colon Tregs ex-pressed RORgt (Fig. 1A). The frequency of RORgt+

Tregs increasedwith age, representingmost intes-tinal Tregs in 1-year-oldmice (fig. S1A). These cellswere not found in the thymus (fig. S1B) and didnot express Helios or Neuropilin-1, markers ofthymically derived Tregs (17, 18), in contrast toconventional RORgt– Tregs (Fig. 1B and fig. S1C).In the colon, most Helios– Tregs were absent inRORgt-deficient mice (Fig. 1B). RORgt+ Tregs also

expressed an activated CD44high CD62Llow phe-notype, as well as increased levels of ICOS, CTLA-4,and the nucleotidases CD39 and CD73 (fig. S1C),altogether indicating robust regulatory functions.Another major subset of intestinal Tregs expressesGata3, responds to IL-33, and is involved in theregulation of effector T cells during inflammation(19, 20). Gata3+ Tregs were distinct from RORgt+

Tregs and expressed Helios, as well as lower levelsof IL-10 (fig. S2).RORgt+ Tregs were profoundly reduced in germ-

free or antibiotic-treated mice, whereas Helios+

and Gata3+ Tregs were unaffected (Fig. 1C and fig.S3). Recolonization of germfree mice with a spe-cific pathogen–free (SPF) microbiota restorednormal numbers of RORgt+ Tregs (fig. S4). Fur-thermore, a consortium of symbionts composedof 17 Clostridia species efficiently induces the gen-eration of Tregs expressing IL-10 in the colon (21),the majority of which expressed RORgt (Fig. 1D).The microbiota has been shown to induce thegeneration of intestinal Tregs through short-chainfatty acids (SCFA) (22, 23) and antigen (11). Wefound that the SCFA butyrate induced an increase

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1Institut Pasteur, Microenvironment and Immunity Unit,75724 Paris, France. 2Laboratory of ExperimentalImmunology, Immunology Frontier Research Center, OsakaUniversity, Suita 565-0871, Japan. 3RIKEN Center forIntegrative Medical Sciences (IMS-RCAI), Yokohama,Kanagawa 230-0045, Japan. 4PRESTO, Japan Science andTechnology Agency, Saitama 332-0012, Japan. 5The Instituteof Medical Science, The University of Tokyo, Tokyo108-8639, Japan. 6INSERM, U1163, Laboratory of IntestinalImmunity, Paris, France. 7Université Paris Descartes-Sorbonne Paris Cité and Institut Imagine, Paris, France.8INRA Micalis UMR1319, Jouy-en-Josas, France. 9Center ofAllergy and Environment (ZAUM), Technische Universität andHelmholtz Zentrum München, Munich, Germany. 10ResearchInstitute of Molecular Pathology, Vienna Biocenter, 1030Vienna, Austria. 11Institut Pasteur, Biology and Genetics ofBacterial Cell Wall, 75724 Paris, France. 12INSERM, GroupeAvenir, 75015 Paris, France. 13Department of InfectionBiology at the Institute of Clinical Microbiology, Immunologyand Hygiene, University Clinic Erlangen and Friedrich-Alexander University Erlangen-Nuremberg, 91054 Erlangen,Germany. 14CREST, Japan Science and Technology Agency,4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan.15Department of Experimental Pathology, Institute forFrontier Medical Sciences, Kyoto University, Kyoto 606-8507,Japan.*These authors contributed equally to this work. †Present address:Center of Allergy and Environment (ZAUM), Technische Universitätand Helmholtz Zentrum München, Munich, Germany. ‡Presentaddress: INSERM, U1163, Laboratory of Intestinal Immunity, andUniversité Paris Descartes-Sorbonne Paris Cité and InstitutImagine, Paris, France. §Corresponding author. E-mail: gerard.eberl@pasteur.fr

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mainly in RORgt+ Tregs (fig. S5). The generationof RORgt+ Tregs was dependent on dendritic cells(DCs) andmajor histocompatibility complex (MHC)class II (Fig. 1E) and induced by oral antigen(ovalbumin) in transferred naïve CD4+ T cells ex-pressing the OT-II transgenic T cell receptor spe-cific for that antigen (Fig. 1F). Altogether, thesedata show that RORgt+ Tregs are induced by mic-robiota and oral antigen, and that microbiota-induced intestinal Tregs express RORgt.TH17 cellsareefficiently inducedby thepathobiont

segmented filamentous bacteria (SFB) that formscolonies on epithelial cells of the small intestine

(24, 25) and by cytokine signaling pathways in-volving the transcription factor Stat3 (26). Sur-prisingly, RORgt+ Tregs differentiated followingsimilar pathways. RORgt+ Tregs were efficientlyinduced by SFB in the small intestine, even thoughmore TH17 cells were induced in these conditions(Fig. 2A). Furthermore, similar to TH17 cells, innatereceptors of the Toll-like receptor and NOD-likereceptor families were not involved (fig. S6). Incontrast, mice deficient for IL-6 or the p19 sub-unit of IL-23 (encoded by Il23a), both involved inthe induction of TH17 cells (27, 28), developedsignificantly less RORgt+ Tregs (Fig. 2B), whereas

Gata3+ Tregs were increased (fig. S7). In accord-ance with the fact that both cytokines signalthrough the transcription factor Stat3, similarresults were obtained in mice that lack expres-sion of Stat3 in Tregs or RORgt

+ cells (Fig. 2B).If TH17 cells and RORgt+ Tregs are induced

through similar pathways, how then is the de-velopment to TH17 cells or RORgt+ Tregs regu-lated? In cell cultures, the vitamin A metaboliteretinoic acid (RA) promotes the generation ofTregs (29) and of RORgt+ Tregs (15) rather than ofTH17 cells. We now find that feeding mice withvitamin A–deficient food, or treating mice with

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Fig. 1. A majority of colon RORg+ Tcells are microbiota-induced RORgt+

Tregs. (A) Expression of Foxp3 and RORgt by CD4+ T cells in double reportermice. SI, small intestine; Co, colon; LPL, lamina propria lymphocytes. (B) Ex-pression of Helios and RORgt by Tregs (FoxP3

+ CD4+ Tcells) from spleen andcolon lamina propria of littermate control (left) and RORgt-deficient mice(right). Right, frequency of colon Helios– Tregs. n = 4 mice per group. (C) Ex-pression of RORgt and Helios by Tregs in the colon of SPFor germfree (GF) miceand frequencies of RORgt+Helios–Tregs (left) andHelios+ Tregs (middle) in SPFandgermfree mice; n = 4mice per group. Right, frequency of RORgt+ Tregs in thecolon of SPFmice or mice treated from birth with a cocktail of antibiotics;n ≥ 4. (D) Expressionof RORgt and IL-10byTregs and frequencyof RORgt+ Tregs inthe colon of germfree mice (left) or mice recolonized for 3 weeks with a con-

sortium of 17 strains of Clostridia (right); n ≥ 4 mice per group. (E) Expressionof RORgt and Helios by colonic Tregs (left) and frequency of RORgt+ Tregs(middle) inCd11cCre xRosa26Dta (DDC) and littermate control mice, n ≥ 3miceper group. Right, frequencies of RORgt+ Tregs in colon of control or MHC classII–deficientmice; n=4. (F) NaïveCD4+Tcellswere isolated fromCD45.2+OT-IIRag2−/− mice and adoptively transferred into CD45.1+ congenic mice andsubsequently fed for 7 days with 1.5% chicken ovalbumin in the drinking water.Expression of RORgt and Helios in small intestine Tregs (left) and frequency ofRORgt+ Tregs in small intestine and colon (right) in host (CD45.1) and donor(CD45.2) cells; n = 7 mice. Data are representative of at least two independentexperiments. Error bars, mean ± 1 SD; ns, not significant; *P < 0.05; **P < 0.01;***P < 0.001, as calculated by Student’s t test.

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Fig. 2. A proinflammatory pathway plus retinoicacid induceRORgt+Tregs. (A) Expression of RORgtandHelios byTregs in germfreemice andgermfreemice recolonized for 3weekswith SFB.Histogramsshow frequencies of RORgt+ Tregs and ratio of TH17cells versus RORgt+ Tregs. n ≥ 4 mice per group.(B) Frequencies of (Helios–) RORgt+ Tregs in colonof Il6−/−, Il23a−/−, Foxp3Cre x Stat3FL/FL, RORgt Cre xStat3FL/FL, and littermate control mice; n ≥ 3 miceper group. (C) Frequencies of RORgt+ Tregs, TH17cells, Helios+ Tregs, and ratio of TH17 cells versusRORgt+ Tregs in colon of mice fed a control diet ora vitamin A–deficient diet frombirth for 10weeks;n ≥ 3mice per group. Data are representative of atleast two independent experiments. Error bars,mean ± 1 SD; *P < 0.05; ***P < 0.001, as calcu-lated by Student’s t test.

Fig. 3. RORgt+ Tregs regulate type2 immune responses. (A) Expres-sion of Foxp3 and Gata3 by smallintestine CD4+ T cells (left), andfrequency of Gata3+ Tregs (middle)and Gata3+ non-Tregs CD4

+ T cells(right) in Foxp3Cre x Rorc(gt)FL/KO

mice; n = 7 mice per group. (B) Survivalcurve (left) of littermate control mice(straight line) or Foxp3Cre x Rorc(gt)FL/FL

mice (hatched line) treated withoxazolone; n = 7 mice per group. Peri-odic acid-Schiff staining of colons fromthe corresponding mice and ratio ofmice with ulcers (middle) and length ofcolons 3 days after oxazolone challenge(right). (C) Egg burden 14, 24, and31 days after infection with H. polygyrusand production of type 2 cytokines byTcells isolated from mesenteric lymphnodes 21 days after infection in controlmice (filled symbols) or Foxp3Cre xRorc(gt)FL/FL mice (open symbols); n ≥5 mice per group. Data are representa-tive of at least two independent experi-ments. Error bars, ± 1 SD; ns, notsignificant; *P < 0.05; **P < 0.01;****P < 0.0001, as calculated by Stu-dent’s t test or Mann-Whitney test.

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an inhibitor of the RA receptor (RARi), preventedthe development of RORgt+ Tregs but not ofHelios

+

Tregs or TH17 cells (Fig. 2C and fig. S8). RA alsopromotes the expansion of ILC3s over ILC2s (30)and the maturation of fetal ILC3s through RAR-mediated regulation of the Rorc locus encodingRORgt (31). Thus, vitaminAmetabolismpromotesthe development of RORgt+ cells and type 3 im-munity, yet favors the development of RORgt+

Tregs over TH17 cells, presumably to limit the num-ber of proinflammatory cells present in thehealthy intestine.We next assessed whether RORgt+ Tregs regu-

late type 2 responses. Mice that lack only RORgt+

Tregs were generated through a conditional knock-out ofRorc in Foxp3+ cells. Such Foxp3Cre x Rorc(gt)FL mice developed increased frequencies ofGata3+ T cells andGata3+ Tregs (Fig. 3A), and, as aconsequence, T cells produced higher amountsof the type 2 cytokines IL-4 and IL-5 (fig. S9A).These mice developed a more severe and lethalform of oxazolone-induced colitis, a model ofulcerative colitis dependent on the type 2 cy-tokines IL-4 (32) and IL-13 (33), as comparedwith their wild-type littermates (Fig. 3B). Incontrast, they were more resistant to infectionby the helminthHeligmosomoides polygyrus, be-cause they produced higher levels of IL-4, IL-5,and IL-13 during the infection (Fig. 3C). TH17cells contributed to the control of type 2 responses,because amore pronounced increase in TH2 cellswas observed in full RORgt-deficient mice (fig.

S9B). Furthermore,RORgt-deficientmiceexpressedhigh levels of immunoglobulin E (IgE) (fig. S9C),a hallmark of type 2 immunity, at levels some-times similar to those found in germfree mice (3).In contrast, Foxp3Cre x Rorc(gt)FL mice did notdevelop increased TH17 or TH1 responses, evenduring acute intestinal inflammation induced bysodium dextran sulfate (fig. S10). These dataare in agreement with the spontaneous type 2pathologies observed in mice lacking extrathy-mically generatedTregs (12) and indicate thatmicro-biota regulate type 2 responses through RORgt+

Tregs and more generally RORgt+ T cells.What are the mechanisms by which RORgt+

Tregs regulate type 2 immunity? We find thatboth cell-intrinsic and cell-extrinsic mechanismsof regulation are involved. Naïve OT-II+ CD4+

T cells developed into RORgt+ Tregs when trans-ferred intomice fed ovalbumin (Fig. 1F), whereasa majority of them developed into Gata3+ Tregswhen cells deficient in RORgt were transferred(Fig. 4A and fig. S11A). However, host Tregs werenot affected, showing that a loss in RORgt affectsTregs only through a cell-intrinsic pathway. Incontrast, the transfer of RORgt-deficient OT-II+

cells affected the generation of both donor andhost TH2 cells, but not TH17 cells, showing thatRORgt+ Tregs regulate TH2 cells also through acell-extrinsic pathway. Furthermore, in RORgt+

Tregs that lacked Stat3, the expression of Gata3was deregulated, and thus both transcription fac-tors were coexpressed (Fig. 4B). This is in accord-

ance with earlier data showing that IL-23 blocksthe IL-33–mediated accumulation of Gata3+ Tregs(20), and, conversely, that the absence of Gata3leads to the expansion of RORgt+ Tregs (19, 34).This cross-inhibition may act directly, as Foxp3binds to Gata3, Stat3 (35), and RORgt (15, 36),and Gata3 binds to the Rorc promoter (19). Incontrast, the expression levels of Gata3 remainedunchanged in Stat3-deficient TH17 cells (fig. S11B).We next investigated the mechanisms of the

cell-extrinsic regulation of TH2 cells by RORgt+

Tregs. Because RORgt+ Tregs express high levels of

IL-10 (Fig. 1D and fig. S2B) (15), we assessedwhether RORgt+ Tregs regulate TH2 cells throughIL-10, the receptor of which activates Stat3 (37).However, IL-10–deficient mice showed massiveexpansion of TH17 cells but no expansion ofGata3+ T cells (fig. S12). RORgt+ Tregs also expresshigh levels of CTLA4 (fig. S1C), shown to regulatethe expression of CD80 and CD86 on DCs (38).As a consequence, the expression of both CD80and CD86 by intestinal DCs was increased inFoxp3Cre x Rorc(gt)FL mice (Fig. 4C). Further-more, in mice that lack CTLA4 expression inTregs, Gata3

+ T cells were significantly expandedin the intestine, whereas TH1 and TH17 cells werenot (Fig. 4D), and serum levels of IgE were in-creased (38). These data indicate that RORgt+

Tregs regulate coactivator functions of DCs throughCTLA4 and thereby regulate the generation ofTH2 cells in the intestine. Finally, RORgt+ Tregsexpress high levels of interferon regulatory factor

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Fig. 4. Mechanisms of regulation by RORgt+ Tregs. (A) Naïve CD4+ T cellswere isolated from CD45.2+ OT-II wild-type or CD45.2+ OT-II RORgt −/− mice,adoptively transferred into CD45.1+ congenic mice, and subsequently fed for7 days with 1.5% chicken ovalbumin. Frequency of Gata3+ Tregs and TH2 cellsin the small intestine in host (CD45.1) and donor (CD45.2) cells; n = 3mice. (B) Expression of RORgt and Gata3 by Tregs in colon of littermatecontrol mice and of Foxp3Cre x Stat3FL/FL mice. (C) Mean fluorescence intensity(MFI) of CD80 and CD86 on colon DCs; n ≥ 5 mice per group. (D) Frequency ofGata3+, RORgt+, or T-bet+ (a marker for TH1 cells) non-Treg CD4+ T cells in

littermate control mice (filled symbols) or Foxp3Cre x CTLA-4FL/FL mice (opensymbols); n = 6 mice per group. (E) Expression of IRF4 protein and transcriptsby RORgt+ Tregs and RORgt– Tregs (gray filled histogram representseffector T cells); n = 3 mice (left) or in triplicates (right). (F) Expression oftranscripts for IL-33, IL-6, and IL-23 in the ileum of SPF and germfree mice, asdetermined by quantitative reverse transcriptase polymerase chain reaction;n = 3 mice per group. Data are representative of at least two independentexperiments. Error bars, ± 1 SD; ns, not significant; *P < 0.05 [0.06 in (A)]; **P <0.01, as calculated by Student’s t test or Mann-Whitney test.

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4 (IRF4) (Fig. 4E), which endows Tregs with theability to suppress TH2 responses (39).Type 2 responses are proposed to perform

“housekeeping” repair functions co-opted for de-fense against large parasites (40). In germfreemice, type 2 immunity is exacerbated (3–6), pos-sibly as a consequence of deregulated repair re-sponses. In accordance with this view, expressionof the type 2 cytokine IL-33 by epithelial cells isincreased in germfree mice (Fig. 4F and fig. S11C).IL-33 promotes the accumulation and functionof microbiota-independent (fig. S3) Gata3+ Tregs,which express high levels of amphiregulin, anepidermal growth factor receptor ligand involvedin tissue repair (20). In contrast, the microbiotainduces type 3 responses through cytokines suchas IL-6 and IL-23 (Fig. 4F) and thereby suppressesthe default type 2 responses (Fig. 3 and fig. S13).A model of the immune system may therefore

be proposed in which type 1, 2, and 3 responses,induced by intracellular threats, tissue injury,and extracellular threats, respectively, establish ahealthy equilibrium. In that model, Treg subsetsare part of each type of responses and play anessential role in balancing the number of effectorsthat are generated during steady state, infection,or injury. As we have evolved and developed inthe presence ofmicrobes, an absence ofmicrobesleads to a loss in type 1 (41) and type 3 responsesand, therefore, to deregulated type 2 responsesassociated with profibrotic and proallergic pathol-ogies (42). A similar mechanismmay account forthe increase, in industrialized nations, of auto-immune pathologies associated with type 3 im-munity (1).

REFERENCES AND NOTES

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ACKNOWLEDGMENTS

We thank B. Ryffel for providing the Il23a−/− mice, I. Förster for theStat3FL/FL mice, and C. Leclerc for Il10−/− mice. We thankL. Polomack for technical assistance and the members of theMicroenvironment and Immunity Unit for discussion and support.The data presented in this manuscript are tabulated in the mainpaper and in the supplementary materials. This work wassupported by the Institut Pasteur, grants from the Agence

Nationale de la Recherche (ANR 11 BSV3 020 01), the Fondation dela Recherche Medicale (DEq. 2010318246), the Fondation Simonee Cino Del Duca from the Institut de France, and an ExcellenceGrant from the European Commission (MEXT-CT-2006-042374).This study has received funding from the French government’sInvestissement d’Avenir program, Laboratoire d’Excellence“Integrative Biology of Emerging Infectious Diseases” (grant no.ANR-10-LABX-62-IBEID). C.O. was supported by a EuropeanMolecular Biology Organization fellowship, S.C. by a Marie Curieintra-European fellowship from the European Union, J.B.W. byJapan Society for the Promotion of Science Young Scientist Bgrant 15K19129, and M.B. by Boehringer Ingelheim.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/349/6251/989/suppl/DC1Materials and MethodsFigs. S1 to S13References (43–53)

16 January 2015; accepted 23 June 2015Published online 9 July 201510.1126/science.aac4263

MUCOSAL IMMUNOLOGY

Individual intestinal symbiontsinduce a distinct population of RORg+

regulatory T cellsEsen Sefik,1 Naama Geva-Zatorsky,1 Sungwhan Oh,1 Liza Konnikova,3 David Zemmour,1

Abigail Manson McGuire,4 Dalia Burzyn,1* Adriana Ortiz-Lopez,1 Mercedes Lobera,5

Jianfei Yang,5 Shomir Ghosh,5 Ashlee Earl,4 Scott B. Snapper,3 Ray Jupp,6

Dennis Kasper,1 Diane Mathis,1,2† Christophe Benoist1,2†

T regulatory cells that express the transcription factor Foxp3 (Foxp3+ Tregs) promote tissuehomeostasis in several settings. We now report that symbiotic members of the humangut microbiota induce a distinct Treg population in the mouse colon, which constrains immuno-inflammatory responses. This induction—which we find to map to a broad, but specific,array of individual bacterial species—requires the transcription factor Rorg, paradoxically, inthat Rorg is thought to antagonize FoxP3 and to promote T helper 17 (TH17) cell differentiation.Rorg’s transcriptional footprint differs in colonic Tregs and TH17 cells and controls importanteffector molecules. Rorg, and the Tregs that express it, contribute substantially to regulatingcolonic TH1/TH17 inflammation. Thus, the marked context-specificity of Rorg results invery different outcomes even in closely related cell types.

FoxP3 regulatoryT (Foxp3+Treg) cells are essen-tial regulators of immunologic homeostasisand responses (1). Beyond their well-describedrole in regulating the activity of other immu-nocytes, Tregs located in parenchymal tissues

control other, nonimmunological, processes. These“tissue Tregs” include those that reside in visceraladipose tissue and regulate metabolic parame-

ters (2, 3) and those that help channel inflam-matory and regenerative events in injuredmuscle(4). The activities, transcriptomes, and T cell re-ceptor (TCR) repertoires of these tissue Tregs aredistinct from their counterparts in secondarylymphoid organs.Another essential and specific population of

tissue Tregs resides in the lamina propria (LP) ofthe digestive tract, in particular in the colon,where these cells modulate responses to com-mensal microbes [reviewed in (5)]. Colonic Tregsare an unusual population that has provokedsome contradictory observations. TCRs expressedby colonic Tregs show marked reactivity againstmicrobial antigens that seem to be importantdrivers of their differentiation and/or expan-sion (6, 7). Many of them appear to arise byconversion from FoxP3– conventional CD4+ Tcells (Tconv) (6, 7), although arguments for a

SCIENCE sciencemag.org 28 AUGUST 2015 • VOL 349 ISSUE 6251 993

1Division of Immunology, Department of Microbiology andImmunobiology, Harvard Medical School, Boston, MA 02115, USA.2Evergrande Center for Immunologic Diseases, Harvard MedicalSchool and Brigham and Women’s Hospital, Boston, MA 02115,USA. 3Division of Gastroenterology and Hepatology, Brigham andWomen's Hospital, Boston, MA 02115, USA, and Department ofMedicine, Harvard Medical School, Boston, MA 02115, USA. 4BroadInstitute of MIT and Harvard, Cambridge, MA 02142, USA.5Tempero Pharmaceuticals, a GSK Company, Cambridge, MA02139, USA. 6UCB Pharma, Slough, Berkshire, UK.*Present address: Jounce Therapeutics, Inc., Cambridge, MA 02138, USA.†Corresponding author. E-mail: cbdm@hms.harvard.edu (D.M.; C.B.)

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T cells+tγThe microbiota regulates type 2 immunity through ROR

Voehringer, Koji Hase, Kenya Honda, Shimon Sakaguchi and Gérard EberlRute Marques, Sophie Dulauroy, Maria Fedoseeva, Meinrad Busslinger, Nadine Cerf-Bensussan, Ivo G. Boneca, David Caspar Ohnmacht, Joo-Hong Park, Sascha Cording, James B. Wing, Koji Atarashi, Yuuki Obata, Valérie Gaboriau-Routhiau,

originally published online July 9, 2015DOI: 10.1126/science.aac4263 (6251), 989-993.349Science 

, this issue pp. 989 and 993Scienceassociated with allergies (see the Perspective by Hegazy and Powrie).

moleculesfactors exhibited enhanced susceptibility to colonic inflammation and had elevated amounts of proinflammatory expressing regulatory T cells that helped maintain immune homeostasis. Mice engineered to lack these transcription

−which required gut microbiota to survive. Multiple bacterial species of the microbiota could induce transcription factor characterized a population of gut regulatory T cells in mice, et al. and Sefik et al.and its microbial contents. Ohnmacht

species of bacteria induce a type of T cell that promotes tolerance between the hostClostridiumin the gut. For instance, Our guts harbor trillions of microbial inhabitants, some of which regulate the types of immune cells that are present

Gut microbes make T cells keep the peace

ARTICLE TOOLS http://science.sciencemag.org/content/349/6251/989

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2015/07/08/science.aac4263.DC1

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http://stm.sciencemag.org/content/scitransmed/7/276/276ra24.fullhttp://stm.sciencemag.org/content/scitransmed/7/271/271ps1.fullhttp://stke.sciencemag.org/content/sigtrans/4/165/mr4.fullhttp://stm.sciencemag.org/content/scitransmed/6/263/263ra158.fullhttp://stm.sciencemag.org/content/scitransmed/7/295/295ed8.fullhttp://science.sciencemag.org/content/sci/349/6251/929.full

REFERENCES

http://science.sciencemag.org/content/349/6251/989#BIBLThis article cites 53 articles, 18 of which you can access for free

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